Date Available
12-14-2011
Year of Publication
2009
Degree Name
Doctor of Philosophy (PhD)
Document Type
Dissertation
College
Medicine
Department
Physiology
First Advisor
Dr. Steven Estus
Abstract
Low density lipoprotein receptor (LDLR) is an apolipoprotein E (apoE) receptor and may play a role in Alzheimer’s disease (AD) development. A single nucleotide polymorphism (SNP), rs688, that has been identified to modulate the splicing efficiency of LDLR exon 12 and is associated with higher cholesterol and AD in some case-control populations. The exon 12 deleted mRNA is predicted to produce a soluble form of LDLR that fails to mediate apoE uptake. To gain additional insights, in this study, I seek to understand the regulation of LDLR splicing efficiency. To identify functional cis-elements within LDLR exon 12, I mutated several conserved putative exonic splicing enhancers (ESEs) to neutralize their affinity to serine/arginine-rich (SR) proteins. Transfection of wild type (WT) or mutant LDLR minigenes in HepG2 cells was performed, and splicing efficiency evaluated by quantitative RT-PCR. The results showed that two functional ESEs within exon 12, near rs688, are critical to LDLR splicing. To identify splicing factors that modulate exon 12 splicing, I co-transfected an LDLR minigene and vectors encoding different SR proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs). After quantifying the splicing efficiency, I found that SRp20 and SRp38 increased exon 11- skipping. Moreover, ectopic expression of SRp38-2 and hnRNP G increased exon 11&12-skipping. Interestingly, the actions of hnRNP G did not require its RNA recognition motif (RRM). To further investigate the role of theses splicing factors on LDLR splicing, I quantified the expression level of these splicing factors as well as LDLR splicing efficiency in human brain and liver. I found that SRp38 mRNA expression is associated with LDLR splicing efficiency. In conclusion, this study discovered that rs688 is located close to the two functional ESEs within LDLR exon 12, and revealed a role of SRp38 in LDLR splicing efficiency.
Recommended Citation
Ling, I-Fang, "REGULATION OF LOW DENSITY LIPOPROTEIN RECEPTOR SPLICING EFFICIENCY" (2009). University of Kentucky Doctoral Dissertations. 794.
https://uknowledge.uky.edu/gradschool_diss/794
